| BackgroundDiabetic nephropathy(DN)is one of devastating microvascular complication of diabetes mellitus and the most common cause of end-stage renal disease(ESRD),with30–40%mortality.DN is characterized by specific renal structure and functional alterations such as glomerular hyperfiltration,microalbuminuria,thickening of the glomerular basement membrane,interstitial fibrosis,and hypertrophy of mesangial cells.Despite current pharmacological treatments,including strategies for optimizing glycemic control and inhibitors of the renin-angiotensin system,these conventional treatments provide incomplete kidney protection.Hence,there is an urgent need for novel therapeutic approaches that efficiently delay the disease progression.The initiating mechanisms underlying the development and progression of renal injury in DN are not well understood,but current knowledge indicates that its pathogenesis is multifactorial.Compelling and increasing evidence has clearly demonstrated that immunity and inflammation play a paramount role in the pathogenesis of DN.Indeed,DN is associated with both systemic and local renal inflammation with the participation of crucial inflammatory cells such as macrophages.In response to various signals,macrophages may undergo classical M1 activation or alternative M2 activation.The M1 phenotype is characterized by the expression of high levels of proinflammatory cytokines,high production of oxygen intermediates,and promotion of Th1 response.In contrast,M2 macrophages are considered to be involved in immunomodulation and promotion of tissue remodeling.With the advent of cell therapy,mesenchymal stem cells(MSCs)are considered as the most attractive cell source for regenerative medicine and provide a promising strategy to against DN.MSCs are self-renewing and multipotent progenitors that can differentiate into a variety of cell types.MSCs can be obtained from various tissues,including bone marrow,skeletal muscle,dental pulp,adipose tissue and umbilical cord.Of note,human umbilical cord-derived MSCs(UC-MSCs)are much younger and lower immunogenic,and have a higher yield without ethical issues and invasive procedures.Thus,human UC-MSCs are considered to be a better choice for clinical applications compared to many other MSCs.Growing evidence in recent years has revealed that MSCs have multiple biological functions,including immunomodulation,anti-inflammation,anti-apoptosis and anti-fibrosis.Because of these characteristics,MSCs have been applied in various diseases,such as respiratory diseases,circulatory system disease,nervous system disease,and urinary system disease.However,the underlying mechanisms of these beneficial effects are not completely elucidated.MicroRNAs(miRNAs)are a class of noncoding single-stranded small RNA with20-22 nucleotides in length.MiRNAs exert a regulatory effect on a wide range of biological cell processes including cell apoptosis,proliferation,and inflammation by incompletely pairing with the 3’-untranslated region of the target m RNA.Previous studies have implicated that MSCs-derived miRNA negatively regulate expression of target genes in diverse biological processes.However,it remains unclear whether and how MSCs-derived miRNA may regulate macrophage polarization in the pathogenesis of DN.Methods1.Determine whether the isolated UC-MSCs meet the standards set by the International Society for Cellular Therapy for MSCs by inducing differentiation and detecting cell surface markers.2.DN was induced in rats by intraperitoneal injection of high doses of streptozotocin, and UC-MSCs(2x10~6/500μL PBS,once a week,twice in total)were injected into DN rats via the tail vein.Renal function and renal pathological changes were used to evaluate the therapeutic effect of UC-MSCs on DN rats.3.The expression and concentration of inflammatory cytokines in the kidney and serum of experimental rats were detected by q RT-PCR and ELISA,respectively; inflammatory cell infiltration and macrophage polarization in the kidney were detected by immunohistochemistry and immunofluorescence,respectively.The regulatory effect of UC-MSCs on macrophage polarization was assessed by co- culturing macrophages with conditioned medium derived from UC-MSCs.4.Through high-throughput micro RNA sequencing,the differentially expressed miRNAs were compared in the kidneys of normal rats and DN rats,as well as in exosomes derived from HEK293T and UC-MSCs..5.Through the bioinformatics platform and dual luciferase reporter system to determine the downstream target genes of the identified miRNA,q RT-PCR and Western Blot were used to explore the downstream pathways.6.miRNA-modified UC-MSCs were injected into DN rats to observe the repair ability on renal injury in DN rats.Results1.The isolated UC-MSCs 1)have the typical growth characteristics of MSCs;2)can positively express CD70,CD90 and CD105,but do not express markers such as CD19,CD34,CD45 and HLA-DR;3)can be induced to differentiate into adipose, osteogenic and chondrogenic cells,which conforms to the standard of MSCs formulated by the International Association for Cell Therapy.2.UC-MSC administration reduces serum creatinine,urea nitrogen and urine protein in DN rats;and attenuates renal histopathological damage such as tubular dilatation, glomerulosclerosis,renal interstitial fibrosis and inflammatory cell infiltration in the kidney of DN rats.3.UC-MSC administration inhibits the expression of inflammatory cytokines such as IL-1β,IL-6 as well as TNF-αand promote the expression of IL-10 in the kidney of DN rats.In addition,UC-MSCs effectively prevents the infiltration of neutrophils and macrophages in the kidneys of DN rats.4.UC-MSCs reduces iNOS expression and promotes ARG1 expression in macrophages,promoting macrophage polarization toward the anti-inflammatory M2 phenotype in vivo and in vitro.5.The expression of miR-146a-5p was decreased in the kidney of DN rats,and significantly increased after UC-MSC administration.In addition,miR-146a-5p is abundant in UC-MSCs-derived exosomes and is closely related to DN kidney injury6.miR-146a-5p reduces the expression of M1 macrophage markers and corresponding inflammatory cytokines,and promotes the expression of M2 macrophage markers and anti-inflammatory cytokines by targeting the TRAF6/STAT1 signaling pathway.7.Compared with UC-MSCs,miR-146a-5p-modified UC-MSC administration further 1)reduces serum creatinine,blood urea nitrogen,urinary protein and repair damaged kidney tissue in DN rats;2)inhibits the infiltration of inflammatory cells and the concentration of inflammatory cytokines in the kidneys of DN rats;3)and promotes the polarization of macrophages to M2 type in DN kidney.ConclusionIn conclusion,our findings demonstrate a potential beneficial effect of UC-MSC administration on the STZ-induced DN.We identify a role of miR-146a-5p/TRAF6signaling in controlling macrophage polarization in UC-MSC-mediated immune regulation.Importantly,the present study suggests that miR-146a-5p-modified UC-MSCs enhance protection against renal injury in DN through facilitating M2macrophage polarization by targeting TRAF6-STAT1 signaling,which may offer new therapeutic approaches for DN currently lacking effective treatment. |
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